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Multidimensional proteomics analysis of amniotic fluid to provide insight into the mechanisms of idiopathic preterm birth.

Buhimschi IA, Zhao G, Rosenberg VA, Abdel-Razeq S, Thung S, Buhimschi CS - PLoS ONE (2008)

Bottom Line: Women displaying the Q-profile (mean+/-SD, gestational age: 25+/-4 weeks, n = 40) were more likely to deliver preterm despite expectant management in the context of intact membranes and normal amniotic fluid clinical results.Utilizing identification-centered proteomics techniques (fluorescence two-dimensional differential gel electrophoresis, robotic tryptic digestion and mass spectrometry) coupled with Protein ANalysis THrough Evolutionary Relationships (PANTHER) ontological classifications, we determined that in amniotic fluids with Q-profile the differentially expressed proteins are primarily involved in non-inflammatory biological processes such as protein metabolism, signal transduction and transport.The altered proteins may offer opportunities for therapeutical intervention and future drug development to prevent prematurity.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut, United States of America. irina.buhimschi@yale.edu

ABSTRACT

Background: Though recent advancement in proteomics has provided a novel perspective on several distinct pathogenetic mechanisms leading to preterm birth (inflammation, bleeding), the etiology of most preterm births still remains elusive. We conducted a multidimensional proteomic analysis of the amniotic fluid to identify pathways related to preterm birth in the absence of inflammation or bleeding.

Methodology/principal findings: A proteomic fingerprint was generated from fresh amniotic fluid using surface-enhanced laser desorbtion ionization time of flight (SELDI-TOF) mass spectrometry in a total of 286 consecutive samples retrieved from women who presented with signs or symptoms of preterm labor or preterm premature rupture of the membranes. Inflammation and/or bleeding proteomic patterns were detected in 32% (92/286) of the SELDI tracings. In the remaining tracings, a hierarchical algorithm was applied based on descriptors quantifying similarity/dissimilarity among proteomic fingerprints. This allowed identification of a novel profile (Q-profile) based on the presence of 5 SELDI peaks in the 10-12.5 kDa mass area. Women displaying the Q-profile (mean+/-SD, gestational age: 25+/-4 weeks, n = 40) were more likely to deliver preterm despite expectant management in the context of intact membranes and normal amniotic fluid clinical results. Utilizing identification-centered proteomics techniques (fluorescence two-dimensional differential gel electrophoresis, robotic tryptic digestion and mass spectrometry) coupled with Protein ANalysis THrough Evolutionary Relationships (PANTHER) ontological classifications, we determined that in amniotic fluids with Q-profile the differentially expressed proteins are primarily involved in non-inflammatory biological processes such as protein metabolism, signal transduction and transport.

Conclusion/significance: Proteomic profiling of amniotic fluid coupled with non-hierarchical bioinformatics algorithms identified a subgroup of patients at risk for preterm birth in the absence of intra-amniotic inflammation or bleeding, suggesting a novel pathogenetic pathway leading to preterm birth. The altered proteins may offer opportunities for therapeutical intervention and future drug development to prevent prematurity.

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Proteomic patterns characteristic for intra-amniotic inflammation and bleeding.Three hypervariable areas of 4 representative SELDI tracings of amniotic fluid (A: 3–4 kDa, B: 10–12.5 kDa and C: 14–17 kDa) are shown. These areas contain the proteomic patterns characteristic of intra-amniotic inflammation (MR score composed of four protein biomarkers: P1: neutrophil defensin-2: P2: neutrophil defensin-1: 3448.09 Da, P3: calgranulin C, P4: calgranulin A), intra-amniotic bleeding (Hb chains) or both (bottom tracins). The x-axis of the tracings represents the molecular weight in Daltons; the y-axis represents the normalized peak intensity. R denotes a reference protein peak present in all fluid samples which corresponds to a fragment of beta-2 microglobulin.
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pone-0002049-g001: Proteomic patterns characteristic for intra-amniotic inflammation and bleeding.Three hypervariable areas of 4 representative SELDI tracings of amniotic fluid (A: 3–4 kDa, B: 10–12.5 kDa and C: 14–17 kDa) are shown. These areas contain the proteomic patterns characteristic of intra-amniotic inflammation (MR score composed of four protein biomarkers: P1: neutrophil defensin-2: P2: neutrophil defensin-1: 3448.09 Da, P3: calgranulin C, P4: calgranulin A), intra-amniotic bleeding (Hb chains) or both (bottom tracins). The x-axis of the tracings represents the molecular weight in Daltons; the y-axis represents the normalized peak intensity. R denotes a reference protein peak present in all fluid samples which corresponds to a fragment of beta-2 microglobulin.

Mentions: We used surface-enhanced laser desorbtion ionization time-of-flight (SELDI-TOF) mass spectrometry to screen for the presence of biomarkers characteristic of inflammation and bleeding in fresh samples of amniotic fluid. The results were not made available to the clinical management team. The methodology for generation of the MR score and identification of bleeding biomarkers has been previously described [5], [6]. Briefly, 5 microliters (µL) of 10-fold diluted amniotic fluid in phosphate-buffered saline solution (PBS) was placed on spots of duplicate H4 arrays (8-spot H4 array; Ciphergen Biosystems). After 1-hour incubation, the sample was aspirated, and the spots individually washed with 20% aqueous acetonitrile solution, air-dried, and covered with energy absorbing molecule (EAM) matrix solution of either 1-µL of 20% saturated α-cyano-4-hydroxycinnamic acid (CHCA) in 0.5% trifluoroacetic acid/50% acetonitrile on one array, or 2 sequential applications of 1-µL 50% saturated solution (in 0.5% trifluoroacetic acid/50% acetonitrile) of sinapinic acid (SPA) on the other. The proteomic arrays were read in the the ProteinChip Reader (model PBS II; Ciphergen Biosystems) using the ProteinChip software (version 3.0; Ciphergen Biosystems). For identification of proteomic profiles characteristic for inflammation (the MR score) and bleeding (Hb peaks), examination of SELDI tracings was targeted in 3 mass ranges: 3000 to 4000 Daltons (Da) (CHCA), 10 to 12.5 kiloDaltons (kDa) (SPA), 14 to 17 kDa (SPA). The data for the biomarkers of the MR score were extracted from the 3000 to 4000 Da (P1 and P2, Figure 1A) and 10 to 12.5 kDa (P3 and P4, Figure 1B) ranges. The data for the Hb peaks were extracted from the 14 to 17 kDa region (Figure 1C). Both the MR score and Hb profile provide qualitative information regarding the presence or absence of intra-amniotic inflammation or bleeding, respectively [5], [6]. A categorical value of 1 is assigned if a biomarker peak is present and 0 if absent. Thus, the MR score (inflammation) ranges from 0 to 4, depending upon the presence or absence of each of the four protein biomarkers and is a measure of the severity of intra-amniotic inflammation. MR scores 3–4 are indicative of severe inflammation. The Hb score (ranging from 0–2 depending with the number of free Hb chains identified) indicates presence of intra-amniotic bleeding for any value different than 0 [6]. The generation of the MR and Hb scores for each SELDI tracing was performed by an investigator who was blinded to the origins of the amniotic fluid sample, clinical presentation, clinical outcome or results of the placental histological examination.


Multidimensional proteomics analysis of amniotic fluid to provide insight into the mechanisms of idiopathic preterm birth.

Buhimschi IA, Zhao G, Rosenberg VA, Abdel-Razeq S, Thung S, Buhimschi CS - PLoS ONE (2008)

Proteomic patterns characteristic for intra-amniotic inflammation and bleeding.Three hypervariable areas of 4 representative SELDI tracings of amniotic fluid (A: 3–4 kDa, B: 10–12.5 kDa and C: 14–17 kDa) are shown. These areas contain the proteomic patterns characteristic of intra-amniotic inflammation (MR score composed of four protein biomarkers: P1: neutrophil defensin-2: P2: neutrophil defensin-1: 3448.09 Da, P3: calgranulin C, P4: calgranulin A), intra-amniotic bleeding (Hb chains) or both (bottom tracins). The x-axis of the tracings represents the molecular weight in Daltons; the y-axis represents the normalized peak intensity. R denotes a reference protein peak present in all fluid samples which corresponds to a fragment of beta-2 microglobulin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2315798&req=5

pone-0002049-g001: Proteomic patterns characteristic for intra-amniotic inflammation and bleeding.Three hypervariable areas of 4 representative SELDI tracings of amniotic fluid (A: 3–4 kDa, B: 10–12.5 kDa and C: 14–17 kDa) are shown. These areas contain the proteomic patterns characteristic of intra-amniotic inflammation (MR score composed of four protein biomarkers: P1: neutrophil defensin-2: P2: neutrophil defensin-1: 3448.09 Da, P3: calgranulin C, P4: calgranulin A), intra-amniotic bleeding (Hb chains) or both (bottom tracins). The x-axis of the tracings represents the molecular weight in Daltons; the y-axis represents the normalized peak intensity. R denotes a reference protein peak present in all fluid samples which corresponds to a fragment of beta-2 microglobulin.
Mentions: We used surface-enhanced laser desorbtion ionization time-of-flight (SELDI-TOF) mass spectrometry to screen for the presence of biomarkers characteristic of inflammation and bleeding in fresh samples of amniotic fluid. The results were not made available to the clinical management team. The methodology for generation of the MR score and identification of bleeding biomarkers has been previously described [5], [6]. Briefly, 5 microliters (µL) of 10-fold diluted amniotic fluid in phosphate-buffered saline solution (PBS) was placed on spots of duplicate H4 arrays (8-spot H4 array; Ciphergen Biosystems). After 1-hour incubation, the sample was aspirated, and the spots individually washed with 20% aqueous acetonitrile solution, air-dried, and covered with energy absorbing molecule (EAM) matrix solution of either 1-µL of 20% saturated α-cyano-4-hydroxycinnamic acid (CHCA) in 0.5% trifluoroacetic acid/50% acetonitrile on one array, or 2 sequential applications of 1-µL 50% saturated solution (in 0.5% trifluoroacetic acid/50% acetonitrile) of sinapinic acid (SPA) on the other. The proteomic arrays were read in the the ProteinChip Reader (model PBS II; Ciphergen Biosystems) using the ProteinChip software (version 3.0; Ciphergen Biosystems). For identification of proteomic profiles characteristic for inflammation (the MR score) and bleeding (Hb peaks), examination of SELDI tracings was targeted in 3 mass ranges: 3000 to 4000 Daltons (Da) (CHCA), 10 to 12.5 kiloDaltons (kDa) (SPA), 14 to 17 kDa (SPA). The data for the biomarkers of the MR score were extracted from the 3000 to 4000 Da (P1 and P2, Figure 1A) and 10 to 12.5 kDa (P3 and P4, Figure 1B) ranges. The data for the Hb peaks were extracted from the 14 to 17 kDa region (Figure 1C). Both the MR score and Hb profile provide qualitative information regarding the presence or absence of intra-amniotic inflammation or bleeding, respectively [5], [6]. A categorical value of 1 is assigned if a biomarker peak is present and 0 if absent. Thus, the MR score (inflammation) ranges from 0 to 4, depending upon the presence or absence of each of the four protein biomarkers and is a measure of the severity of intra-amniotic inflammation. MR scores 3–4 are indicative of severe inflammation. The Hb score (ranging from 0–2 depending with the number of free Hb chains identified) indicates presence of intra-amniotic bleeding for any value different than 0 [6]. The generation of the MR and Hb scores for each SELDI tracing was performed by an investigator who was blinded to the origins of the amniotic fluid sample, clinical presentation, clinical outcome or results of the placental histological examination.

Bottom Line: Women displaying the Q-profile (mean+/-SD, gestational age: 25+/-4 weeks, n = 40) were more likely to deliver preterm despite expectant management in the context of intact membranes and normal amniotic fluid clinical results.Utilizing identification-centered proteomics techniques (fluorescence two-dimensional differential gel electrophoresis, robotic tryptic digestion and mass spectrometry) coupled with Protein ANalysis THrough Evolutionary Relationships (PANTHER) ontological classifications, we determined that in amniotic fluids with Q-profile the differentially expressed proteins are primarily involved in non-inflammatory biological processes such as protein metabolism, signal transduction and transport.The altered proteins may offer opportunities for therapeutical intervention and future drug development to prevent prematurity.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut, United States of America. irina.buhimschi@yale.edu

ABSTRACT

Background: Though recent advancement in proteomics has provided a novel perspective on several distinct pathogenetic mechanisms leading to preterm birth (inflammation, bleeding), the etiology of most preterm births still remains elusive. We conducted a multidimensional proteomic analysis of the amniotic fluid to identify pathways related to preterm birth in the absence of inflammation or bleeding.

Methodology/principal findings: A proteomic fingerprint was generated from fresh amniotic fluid using surface-enhanced laser desorbtion ionization time of flight (SELDI-TOF) mass spectrometry in a total of 286 consecutive samples retrieved from women who presented with signs or symptoms of preterm labor or preterm premature rupture of the membranes. Inflammation and/or bleeding proteomic patterns were detected in 32% (92/286) of the SELDI tracings. In the remaining tracings, a hierarchical algorithm was applied based on descriptors quantifying similarity/dissimilarity among proteomic fingerprints. This allowed identification of a novel profile (Q-profile) based on the presence of 5 SELDI peaks in the 10-12.5 kDa mass area. Women displaying the Q-profile (mean+/-SD, gestational age: 25+/-4 weeks, n = 40) were more likely to deliver preterm despite expectant management in the context of intact membranes and normal amniotic fluid clinical results. Utilizing identification-centered proteomics techniques (fluorescence two-dimensional differential gel electrophoresis, robotic tryptic digestion and mass spectrometry) coupled with Protein ANalysis THrough Evolutionary Relationships (PANTHER) ontological classifications, we determined that in amniotic fluids with Q-profile the differentially expressed proteins are primarily involved in non-inflammatory biological processes such as protein metabolism, signal transduction and transport.

Conclusion/significance: Proteomic profiling of amniotic fluid coupled with non-hierarchical bioinformatics algorithms identified a subgroup of patients at risk for preterm birth in the absence of intra-amniotic inflammation or bleeding, suggesting a novel pathogenetic pathway leading to preterm birth. The altered proteins may offer opportunities for therapeutical intervention and future drug development to prevent prematurity.

Show MeSH
Related in: MedlinePlus